Not applicable
The primary cell wall of plants has been described as a network of cellulose microfibrils embedded in a hemicellulosic polysaccharide matrix, which interacts to some degree with an additional co-extensive matrix of pectin and other less abundant components including structural proteins (Carpita, N. C., et al., Plant J. 3:1 (1993)). In dicotyledons the predominant hemicellulose is xyloglucan and it has been suggested that cellulose microfibrils are coated and tethered by a framework of xyloglucan polymers (Hayashi, T., et al., Plant Physiol. 75:596 (1984); McCann, M. C., et al., J. Cell Sci. 96:323 (1990)). In a turgid cell, disassembly of this potentially load-bearing hemicellulose-cellulose network could provide a rate limiting step to cell wall expansion in elongation of cells, although an enzymic basis for wall loosening remains to be established.
In addition to elongation growth, disassembly of hemicellulose also appears to be integral to cell wall metabolism during fruit ripening in which cells typically undergo a complex change in textural and Theological characteristics. During ripening, both the pectic and hemicellulosic polymers generally undergo substantial depolymerization and solubilization (Gross, K. C., et al., Plant Physiol. 63:117 (1979); Huber, D. J., Hortic. Rev. 5:169 (1983)). Most of the research in the field has focused on pectin degradation, which results from the action of the ripening-related enzyme polygalacturonase (PG), as the key element underlying the softening process. Molecular genetic studies, however, have revealed that this process is not the primary determinant of fruit softening (Smith, C. J. S., et al., Nature 334:724 (1988); Giovannoni, J. J., et al., Plant Cell 1:53 (1989)), but may be a factor in other aspects of fruit quality (Schuch, W. et al., HortScience 26:1517 (1991)). Disassembly of the hemicellulose component of the wall during ripening is common to most fruit although the extent varies between species and most likely reflects the degradation of a mixture of polysaccharides by multiple enzymes. Candidates for mediating hemicellulose modification as a mechanism for cell expansion include endo-1,4-xcex2-glucanases (EGases or xe2x80x9ccellulasesxe2x80x9d) (Fry, S. C., Physiol. Plant. 75:532 (1989)) and xyloglucan endotransglycosylases (XETs) (Fry, S. C., et al., Biochem. J. 282:821 (1992); Nishitani, K., et al., J. Biol. Chem. 267:21058 (1992)), which have both been associated with rapidly expanding tissues. Neither of these classes of enzymes, however, appears to cause extension of isolated cell walls in vitro (McQueen-Mason, S. J., et al., Plant Cell 4:1425 (1992)). Xyloglucan represents the predominant hemicellulose in many fruit including tomato, where degradation is apparent during ripening in wild fruit, but not in fruit of the rin (ripening inhibitor) tomato mutant which softens extremely slowly (Maclachlan, G., et al., Plant Physiol. 105:965 (1994)). Fruit ripening has been associated with both EGases (Lashbrook, C. C., et al.); Gonzalez-Bosch, C., et al., Plant Physiol. 111:1313 (1996)) and XETs (Maclachlan, G., et al., Plant Physiol. 105:965 (1994); Arrowsmith, D. A., et al., Plant Mol. Biol. 28:391 (1995)); however, the importance of these and other as yet uncharacterized enzymes in modifying hemicellulose abundance, distribution and interaction with other cell wall components in fruit have yet to be determined.
A class of proteins called expansins has recently been identified that cause cell wall loosening in stress-relaxation assays but which lack detectable hydrolytic or transglycosylase activity (McQueen-Mason, S. J., et al., (1992); McQueen-Mason, S. J., et al., Planta 190:327 (1993); McQueen-Mason, S. J., Plant Physiol. 107:87 (1995)). It has been proposed that expansins disrupt non-covalent linkages, such as hydrogen bonds, at the cellulose-hemicellulose interface, thereby loosening an important constraint to turgor-driven cell expansion (McQueen-Mason, S. J., (1995)).
Expansin gene families have been identified in cucumber, rice and Arabidopsis (Shcherban, T. Y., et al., Proc. Natl. Acad. Sci. USA 92:9245 (1995)) suggesting that divergent isoforms may act on different components of the cell wall, exhibit differential developmental and environmental regulation or tissue and cell-specific expression. Expansins, to date, have been examined only in vegetative tissues where the action of this class of proteins is to loosen cell walls. There has been no indication that expansins are expressed in fruits. The processes by which expansins contribute to the disassembly of cell walls is not known. Although significant progress has been made in the understanding of fruit ripening, new methods of controlling fruit ripening are needed. The present invention and adaptations of this invention addresses these needs.
The present invention is based, in part, on the isolation and characterization of expansin (Ex1) genes from fruits. The invention provides for isolated nucleic acid molecules comprising a tomato LeEx1 polynucleotide sequence, of about 900-1200 nucleotides and typically about 1100 nucleotides in length, which specifically hybridizes to SEQ ID NO: 1 under stringent conditions. The LeEx1 polynucleotides of the invention encode a LeEx1 polypeptide of about 200-300 amino acids but more typically about 260 amino acids, as shown in SEQ ID NO: 2. In addition, the invention encompasses isolated nucleic acid molecules comprising strawberry FaEx1 polynucleotide sequences, which specifically hybridize to SEQ ID NO: 3 under stringent conditions. In addition to tomato- and strawberry-derived Ex1 polynucleotides and the polypeptides encoded by the polynucleotides, this invention encompasses isolated nucleic acid molecules comprising a CmEx1 polynucleotide sequence from melon, which specifically hybridizes to SEQ ID NO: 5 under stringent conditions.
The nucleic acids of the invention may also comprise expression cassettes containing a plant promoter operably linked to an Ex1 polynucleotide. In some embodiments, the promoter is from a gene active in fruit. The Ex1 polynucleotide may be linked to the promoter in a sense or antisense orientation.
Methods of inhibiting Ex1 expression, and thus modifying cell walls in plant tissues and softening in fruit, in a plant are also provided. The methods comprise introducing into a plant an expression cassette containing a plant promoter operably linked to a Ex1 polynucleotide. The Ex1 may encode a Ex1 polypeptide or may be linked to the promoter in an antisense orientation. The expression cassette can be introduced into the plant by any number of means known in the art, including use of Agrobacterium tumefaciens vector or through sexual reproduction. An example of a polypeptide useful for this purpose is LeEx1 from tomato.
Methods of enhancing Ex1 expression, and thus modifying cell walls in plant tissues and softening in fruit, in a plant are also provided. The methods comprise introducing into a plant an expression cassette containing a plant promoter operably linked to a Ex1 polynucleotide. The Ex1 may encode a Ex1 polypeptide. The expression cassette can be introduced into the plant by any number of means known in the art, including use of Agrobacterium tumefaciens vector or through sexual reproduction.
The promoters of the invention can be used in methods of targeting expression of a desired polynucleotide to fruits or other organs of a plant. The methods comprise introducing into a plant an expression cassette containing a tissue-specific, for example, a fruit ripening-specific, promoter operably linked to a Ex1 polynucleotide sequence.
The invention also provides for transgenic plants comprising an expression cassette containing a plant promoter operably linked to an Ex1 polynucleotide. The Ex1 may encode a Ex1 polypeptide or may be linked to the promoter in an antisense orientation. The plant promoter may be from any number of sources, including a gene typically active in the cells of the fruit of a plant. The transgenic plant can be any desired plant but is often a member of the genera Lycopersicon, Fragaria or Cucumis.